1. Field of the Invention
The present invention relates to a molecular analysis light detection method, such as a molecular fluorescence detection method such as fluorescence correlation spectroscopy or fluorescence intensity distribution analysis for detecting behavior of a substance to be analyzed labeled with a fluorescent label contained in a sample, or Raman spectroscopy for spectroscopically analyzing Raman scattering light from a substance to be analyzed. The present invention also relates to a molecular analysis light detection apparatus for use with the method, and a sample plate.
2. Description of the Related Art
Conventionally, apparatuses for detecting the number and/or mobility of particles or molecules that emit fluorescence by receiving fluorescent signals from a small area and statistically processing the signals have been proposed and used in practice, such as Fluorescence Correlation Spectroscopy (or FCS, see “Fluorescence Correlation Spectroscopy. II. An Experimental Realization”, D. Magde et al., Biopolymers, Vol. 13, pp. 29-61, 1974) and Fluorescence Intensity Distribution Analysis (or FIDA, see International Patent Publication No. WO98/16814). For example, a fluorescence correlation spectroscopy system is commercially available from Wako Pure Chemical Industries, Ltd., and a single-molecule fluorescence analysis system is commercially available from Olympus Corporation.
These apparatuses detect changes in fluorescent molecules moving in and out of a small volume (i.e., fluctuation of fluorescent signals). In conventional apparatuses, a confocal laser is used as an optical system for exciting fluorescence, and a confocal microscope is used as an optical system for detecting the fluorescence, wherein the focal spot of laser light emitted from the confocal laser is reduced to as small as one femtoliter, so that only the fluorescence which is emitted when a molecule is moving through the confocal area is detected with a very high sensitivity. Specifically, the focal spot of the laser light for illumination is reduced, an objective lens having a high numerical aperture (NA) is provided for collecting the fluorescence, and a pinhole is provided at a position (image position) that is conjugate with the focal spot position of the objective lens just before a photodetector, wherein an area in the in-plane (x-y) direction is limited by the objective lens, and the detection area in the optical axis direction (z-axis direction) is further limited by the pinhole to achieve the small measurement volume of one femtoliter.
Small molecules move fast and pass through the confocal area quickly, and thus rapid changes are observed in the intensity of the fluorescence signal. In contrast, large molecules move slowly, and thus slow changes are observed in the intensity of the fluorescence signal. A motion velocity of the molecule can be found from the frequency of fluctuation of the intensity of the fluorescence signal using an autocorrelation technique to estimate the size of the molecule.
As described above, the conventional apparatuses need to be provided with a high-NA objective lens. However, since the objective lens with a NA that is high enough to detect the molecular fluorescence in the small volume is very expensive, the entire apparatus becomes expensive.
With the laser illumination, the illumination area in the in-plane direction can sufficiently be reduced by the high-NA objective lens, however, the illumination area in the optical path direction cannot be limited, and fluorescent bodies that moving in and out of the optical axis are excited. Therefore, it is necessary to provide the pinhole to limit the detection area in the z-axis direction.
As a means for solving the problem associated with use of the laser illumination, apparatuses for detecting behavior of molecules according to fluorescence correlation spectroscopy using evanescent illumination, which excite the fluorescence with an evanescent wave, are disclosed in “Local Diffusion and Concentration of IgG near Planar Membranes: Measurement by Total Internal Reflection with Fluorescence Correlation Spectroscopy”, T. E. Starr and N. L. Thompson, Journal of Physical Chemistry B, Vol. 106, pp. 2365-2371, 2002 and Japanese Unexamined Patent Publication No. 2003-294631.
In these apparatuses, an evanescent wave is generated by applying excitation light so that the excitation light is totally reflected at an interface between a sample and a sample contact surface of a plate that contacts with the sample, and the evanescent wave is used as the illumination for exciting the fluorescence. The evanescent wave reaches only within a range of several hundred nanometers from the interface. Therefore, using this evanescent illumination, the depth of the area for exciting the fluorescence along the optical axis direction (z-axis direction) can be limited without providing the pinhole.
Microscope apparatuses that use the evanescent wave as illumination light for detecting fluorescence have been proposed, for example, in “Single Molecule Imaging of Fluorophores and Enzymatic Reactions Achieved by Objective-Type Total Internal Reflection Fluorescence Microscopy”, Biochemical and Biophysical Research Communications, Vol. 235, pp. 47-53, 1997 and Japanese Unexamined Patent Publication No. 10(1998)-090169. In these microscope apparatuses, however, sufficient sensitivity for allowing the single-molecule measurement is not obtained.
In either of the above-described apparatuses, an objective lens with a high NA (>0.3) is necessary for the detection system. Since the objective lens having the NA high enough to detect the molecular fluorescence in the small volume is very expensive, the entire apparatus becomes expensive.
This is also the case in apparatuses for analyzing molecular-level behavior of a substance to be analyzed that generates Raman scattering light by detecting the Raman scattering light in a small volume.